The KD and Bmax values were determined through an in vitro binding assay. NCCIT embryonal carcinoma cells were plated at 200 000 cells/well in a poly-d -lysine coated clear 12-well plate (Corning, Cat. No. 354470) and allowed to adhere for 24 h at 37 °C and 5% CO2. The cells were either incubated with 0.62–50 nM of [89Zr]Zr-DFO-Bsg or subjected to blocking by coincubation of 0.62–50 nM of [89Zr]Zr-DFO-Bsg with >80-fold excess nonradiolabeled Bsg in triplicate for 1 h at room temperature. Following incubation, the media were removed, and cells were washed twice with 1× PBS, detached with 0.25% trypsin-EDTA, and collected in microcentrifuge tubes. The radioactivity of the samples and concentration standards were measured with a gamma counter (PerkinElmer Wizard2 (link) 2480) and plotted as sites/cell versus antibody concentration (nM). GraphPad Prism was used to determine the KD and Bmax with a one-site association curve.
Wizard2 link 2480
Manufactured by PerkinElmer
Sourced in United States
The Wizard2 2480 is a multi-mode microplate reader designed for research applications. It can detect various types of luminescent, fluorescent, and absorbance signals in microplates. The Wizard2 2480 is capable of performing a range of assays, including cell-based, biochemical, and molecular biology experiments.
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3 protocols using wizard2 link 2480
1
Radiolabeled Bsg Binding Assay
2
Biodistribution Study of Zirconium-89 Labeled Bsg
3
Insulin-Induced Glucose Uptake in Mice
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Eight-week-old and 96-week-old male mice (n = 5, each group) were assigned to the control and insulin-loaded groups. Those in the insulin-loaded groups were intraperitoneally injected with human insulin (2 U/kg body weight, Eli Lilly & Co., Kobe) 30 min prior to 18F-FDG injection. We excluded one of the 96-week-old mice in the insulin-loaded group because the blood glucose level did not decrease after insulin loading. Each animal was initially anesthetized with 4% isoflurane in air and maintained via spontaneous ventilation with 2% isoflurane in air. 18F-FDG (11.5 MBq/0.1 ml) was injected into the tail vein. Ninety minutes later, the animals were sacrificed and their organs were excised. The organs (muscle, heart, lungs, spleen, pancreas, white adipose tissue (superior pole of epididymis), testes, stomach, small intestine, large intestine, kidneys, liver, brown adipose tissue (between the shoulder blades), and brain) and blood samples (blood and plasma) were weighed, and their radioactivity was determined with a gamma counter (WIZARD2 (link) 2480; PerkinElmer, USA). After decay correction, the percentage of injected dose per gram of tissue was obtained and normalized to the animal weight [%ID/g tissue/kg body weight (%ID/g/kg)]. Blood samples for glucose concentration measurement were obtained from the control group and insulin-loaded groups.
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